To compare the influence of different temperatures and curvature radii on the cyclic fatigue resistance of F6 SkyTaper (F6ST) and One Curve (OC) single file nickel-titanium rotary instruments. Methods: A total of 120 instruments of F6ST and OC #25.06 were evaluated in 5 mm and 3 mm curvature radii at two temperatures (20 °C ± 1 °C and 37 °C ± 1 °C) in 16 mm stainless steel artificial canals associated with a curvature of 60°. The cyclic fatigue of tested files was assessed by employing a customized testing apparatus and expressed as times to fracture (TtF). A statistical analysis was performed with the significance level set at 95%. Results: All instruments decreased their TtF at 37 °C except for OC in the 3 mm radius, in which no significant difference was detected between 20 °C and 37 °C. A 3 mm curvature radius negatively affected TtF of all tested instruments, except for F6ST at 20 °C. F6ST had higher TtF than OC in the 3 mm radius at 20 °C, with no significant difference between them in the other tested conditions. Conclusions: Under the limits of the present in vitro study, body temperature impaired cyclic fatigue resistance of all files, except for OC in the 3 mm curvature radius. All instruments exhibited lower times to fracture in the 3 mm radius, excluding F6ST at 20 °C.